Nota Lepi. 42(2) 2019: 137–150 | DOI 10.3897/nl.42.33190 DNA barcoding of Zygaenidae (Lepidoptera): results and perspectives Konstantin A. Efetov1,*, Anna V. Kirsanova1,*, Zoya S. Lazareva1,*, Ekaterina V. Parshkova1,*, Gerhard M. Tarmann2, Rodolphe Rougerie3, Paul D. N. Hebert4 1 V. I. Vernadsky Crimean Federal University, Department of Biological Chemistry and Laboratory of Biotechnology, 295051 Simferopol, Russia; [email protected] 2 Tiroler Landesmuseen, Ferdinandeum, Naturwissenschaftliche Abteilung, Sammlungs- und Forschungszentrum, Krajnc Straße 1, 6060 Hall, Austria; [email protected] 3 Muséum national d’Histoire naturelle, Département Systématique et Evolution, 57 Rue Cuvier, CP50 75005 Paris, France; [email protected] 4 Canadian Centre for DNA Barcoding, Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road, East Guelph, N1G 2W1 Ontario, Canada; [email protected] http://zoobank.org/E54794AA-8565-4DB5-9E29-C30DF598A225 Received 18 January 2019; accepted 2 August 2019; published: 2 October 2019 Subject Editor: Jadranka Rota Abstract. The present study provides a DNA barcode library for the world Zygaenidae (Lepidoptera). This study reports 1031 sequence data of the COI gene DNA barcodes for more than 240 species in four of the five subfamilies of the family Zygaenidae. This is about 20% of the world Zygaenidae species. Our results demonstrate the specificity of the COI gene sequences at the species level in most of the studied Zygaenidae and agree with already established taxonomic opinions. The study confirms the effectiveness of DNA barcod- ing as a tool for determination of most Zygaenidae species. However, some of the results are contradictory. Some cases of shared barcodes have been found, as well as cases of deep intraspecific sequence divergence in species that are well separated by morphological and biological characters. These cases are discussed in detail. Overall, when combined with morphological and biochemical data, as well as biological and ecological observations, DNA barcoding results can be a useful support for taxonomic decisions. Introduction Zygaenidae Latreille, 1809, is a family of Lepidoptera well known for the biochemical properties of its species, capable of synthesizing hydrogen cyanide used as a defensive mechanism. Zygae- nids, commonly known as burnet, forester and smoky moths, are typically day-flying insects. The family encompasses about 1200 species distributed worldwide, of which several are known as pests. Many species have restricted distributions and represent very sensitive ecological indicators, often used, along with butterflies, as an important umbrella group for ecological evaluations (Naza- rov and Efetov 1993; Schmitt 2003; Tarmann 2009). * Disclaimer: Author(s) from Crimea of articles appearing in Nota Lepidopterologica is/are solely responsible for the proper indication of his/her/their actual postal address and affiliation. Nota Lepidopterologica is a scientific journal and cannot be held responsible for political tensions or disputes between governments or persons. 138 Efetov et al.: DNA barcoding of Zygaenidae (Lepidoptera): results and perspectives The taxonomy of these moths is generally well established; it is based on the comparison of morphological characters (habitus and genitalia morphology), but noticeably also in a large part on the integration of biological and ecological characters rarely available (or only available to a lesser extent) in other families of moths. A global taxonomic system was proposed by Alberti (1954, 1958– 1959) in a comprehensive revision of the world’s fauna. This system has been improved during the past 60 years (Naumann and Tremewan 1984; Tarmann 1984, 2004; Efetov and Tarmann 1999, 2012; Hofmann and Tremewan 1996; Yen 2003), based on an exceptional variety of characters such as larval morphology (the chaetotaxy of larvae (Efetov and Hayashi 2008), microstructures of the integument (Efetov and Tarmann 2017a)), head morphology (including biometry), characters in the structure of the antennae, wings, legs, scales, abdomen (e.g. coremata, lateral ‘glands’), special habits of larvae (e.g. leaf mining, boring or free feeding), cocoon construction, special calling and mating habits (Efetov 1996a, 1998a, 1998b, 1999; Efetov and Tarmann 2013b, 2017b; Efetov et al. 2011b; Efetov and Knyazev 2014; Knyazev et al. 2015), pheromones (Subchev et al. 1998, 2013, 2016; Efetov et al. 2010b, 2014b, 2014c, 2015b, 2016, 2018; Razov et al. 2017; Cengiz Can et al. 2018), mimicry, the examination of the karyotypes (Efetov et al. 2004, 2015a), protein electrophoresis results, biochemical analyses combined with the toxicity of the Zygaenidae and the study of antigen properties of haemolymph proteins (monoclonal immunosystematics) (Efetov 2005). This refined system, however, still retains some unsolved questions, especially with respect to phylogenetic relationships. The evolution of the family has been partly investigated over the past decade through the use of molecules, morphology or a combination of both (Efetov 2006, 2012b; Efetov and Savchuk 2009; Efetov et al. 2011a, 2010b, 2014b, 2014c, 2015b, 2016; Subchev et al. 2013, 2016; Efetov and Tarmann 2013b; Mollet 2015), although genetic studies at intraspecific and interspecific levels have been generally limited to species of economic importance (Schmitt and Seitz 2004). The distinction of closely related species often relies on the use of very precise sets of characters and can be highly challenging for non-experts, precluding a broader use of these insects as ecological indicators. The family Zygaenidae has a worldwide distribution and is divided into five subfamilies: Inouelinae Efetov and Tarmann, 2017 (Oriental), Procridinae Boisduval, 1828 (Holarctic, Afro- tropical, Oriental, Australian, Neotropical), Chalcosiinae Walker, 1865 (Palaearctic, Oriental), Callizygaeninae Alberti, 1954 (Oriental), and Zygaeninae Latreille, 1809 (Palaearctic, Oriental, Afrotropical) (Alberti 1954, 1958–1959; Tarmann 1984, 1994, 2004; Efetov and Tarmann 2012, 2017a; Hofmann and Tremewan 1996; Efetov et al. 2014a; Yen 2003). Several molecular phylogenetic studies of Zygaenidae were published by O. Niehuis et al. (2006a, 2006b, 2007). Selected morphological and biological characters were investigated togeth- er with RNA secondary structure variations (Niehuis et al. 2006a) or by the analysis of several mi- tochondrial and nuclear markers (Niehuis et al. 2006b, 2007). These studies are mainly restricted to species of the subfamily Zygaeninae (genus Zygaena Fabricius, 1775) (Niehuis et al. 2006a, 2007). Only a few specimens of Procridinae, Callizygaeninae and Chalcosiinae were included (Niehuis et al. 2006b) while Inouelinae are completely absent. Our project “DNA barcoding of Zygaenidae moths” (ZYGMO) started in 2009 (Efetov et al. 2010) using the COI gene fragment proposed by Hebert et al. (2003a, 2003b) as a standard DNA barcode (Ratnasingham and Hebert 2007) with the goal to initiate a library of DNA barcode se- quences for Zygaenidae species as a new tool for species identification in this family. Moreover, it was expected to confirm known species-complexes and possibly find new ones, as well as so Nota Lepi. 42(2): 137–150 139 far overlooked cryptic diversity. Each Zygaenidae barcode record in the Barcode of Life Data Systems (BOLD, http://www.boldsystems.org) (Ratnasingham and Hebert 2007) is accompanied by specimen images, detailed and geo-referenced collection data, complete taxonomic information and voucher repository data. These activities were undertaken in close cooperation between the Crimean Federal University (Crimea), the Tiroler Landesmuseen, Ferdinandeum (Austria) and the Biodiversity Institute of Ontario at the University of Guelph (Canada) under the framework of the “International Barcode of Life” (iBOL) project. There is another, smaller scale project focused on barcoding Zygaenidae and Papilionoidea, but this recent effort was geographically restricted to Switzerland (Litman et al. 2018). In this paper, we conduct a critical analysis of our Zygaenidae DNA barcoding results. The current taxonomic system for studied species is discussed in light of the relevant results from our sequence analysis combined with traditionally used characters. Some principal remarks on bar- coding and taxonomy as well as contradictory results and examples requiring special attention are listed and discussed below. Materials and methods Specimen sampling for DNA barcoding Zygaenidae specimens from all biogeographical regions of the world were provided by scientists from different countries: К. А. Еfetov (Russia), G. M. Tarmann (Austria), B. Mollet, E. Drouet and J.-M. Desse (France), O. G. Gorbunov and A. N. Zamesov (Russia), I. G. Pljushtch (Ukraine), Th. Keil (Germany), and others (see acknowledgements). The analysed species are listed in Suppl. material 1: Table S1. DNA barcodes were obtained by sampling legs from dry specimens or specimens preserved in 96% ethanol in the following institutions: the Crimean Federal University (Crimea), Tiroler Landesmuseen, Ferdinandeum (Austria), Research collection of Bernard Mollet (France), Re- search collection of Thomas Keil (Germany), Research collection of Eric Drouet (France), Re- search collection of Jean-Marie Desse (France), Schmalhausen Institute of Zoology (Ukraine), Severtsov Institute of Ecology and Evolution of Russian Academy of Sciences (Russia). Sampling was usually restricted to a few specimens per species, with species